The embryonic chick is a widely used model for studying various biological processes. The low cost and easy availability of this system provide major advantages compared to in vivo systems utilizing sentient animals. In addition, many in vivo systems, particularly model systems for studying human tissues and organs, require transplanting an examined graft in an immune compromised animal, in order to avoid graft rejection. Such animals, for example severe combined immune deficient (SCID) mice, are expensive, sickly and hard to maintain. The development of simple, cost-effective systems for mammalian explants is therefore desirable.
The skin is a complex three-dimensional organ that covers the exterior of the body. It is our first line of protection from the myriad microorganisms in our environment, prevents us from dehydrating, and is critically involved in body heat regulation. In addition, a specialized modification of the skin, the hair, is of vital social and psychological importance. Because the skin and hair are very often affected by disease, mechanical and heat damage, there is considerable interest in the mechanisms involved in various physiological or pathophysiological processes associated with human skin and hair. There is a need to research dermatological processes such as the influence of cosmetics and environmental substances on skin, as well as wound healing in the absence and the presence of pharmaceutical and environment substances.
Because the study of human skin in vivo is limited for ethical and medical reasons, most current strategies for the testing of therapies for treating skin damage and disease involve the extensive use of experimental animals. It is common, for example, to investigate the toxicological properties of topically administered materials such as therapeutic formulations or cosmetics by applying the substance to the skin of an experimental animal. Animal studies have also played an important role in preclinical trials of compounds involved in wound healing. Another technique involving the use of experimental animals to study intact human skin is the use of a Non-obese diabetic Severe Combined Immunodeficiency (NOD-SCID) mouse for the long term observation of human skin xenografts.
Animal testing often entails great expense, and involves many legal and ethical issues. Although it is possible to study some properties of human skin cells using cell cultures as disclosed in U.S. Pat. No. 5,512,475 or in vitro micro-organs as disclosed in U.S. Pat. No. 5,888,720, these models require using a population of diffusion nourished skin cells. These models therefore lack important features of skin physiology, such as circulating blood cells that can participate in inflammation and in wound healing. Furthermore, most in-vitro systems require that the epidermis be submerged in medium rather than exposed to air, precluding the possibility of using these systems to conduct experiments with topical treatments such as detergents, cosmetics, radiation, or topically applied pharmaceutical compositions.
It would be advantageous to have a realistic ex vivo model of intact human skin to obviate the need for using experimental animals or recruiting human volunteers. The chick embryo has been used as an in vivo model for the investigation of a number of biological systems for over a century, and has been favored for its simplicity, easy availability, immunodeficient properties, and for the highly vascularized structure of the CAM. For example, the Hen's Egg Test-Chorioallantoic Membrane (HET-CAM) assay is a well-known method for screening the irritancy potential of topically applied compositions such as cosmetics.
The first disclosure of the grafting of human skin upon the chorioallantois of chick embryos was reported by Goodpasture et al. in 1938 (J Exp Med 68:891-904). Goodpasture et al. disclosed the grafting of human and other animal (rabbit, chicken) skin upon the CAM of chick embryos, Goodpasture et al. reported that the epithelium of the chorioallantois fuses with that of the graft, the collagen fibers of the corium interlace with those of the membrane after the separation or disappearance of the ectodermal layer, and the blood vessels of the chick anastomose with those of the graft. This vascular communication between the two tissues is largely responsible for the nourishment of the graft by affording a plasmatic circulation. No laboratory or clinical applications of the system were disclosed by Goodpasture et al. They reported that their human skin grafts may be experimentally infected with viruses, though no details or experimental data were provided.
More recently, after a 60 year period during which there seems to be a dearth of relevant publications, Kunzi-Rapp et al. 1999 (Kunzi-Rapp K, Rück A, Kaufmann R. 1999. Arch Dermatol Res. 291, 290-295) published a study where human skin was grafted to the CAM of chicken eggs. They reported histological and immunohistochemistry results showing the revascularization after 2 to 3 days by reperfusion of pre-existing graft vasculature as well as differentiated human epidermis and dermis containing all cellular and extracellular constituents such as skin immune cells. Based upon these findings, the authors speculated that the CAM system could be used as an alternative system to animal models for studying regulation of cell adhesion molecules, angiogenesis in initial wound healing, and skin toxicology, though none of these were demonstrated.
A modified CAM-based system for investigating wound healing is presented in Roux et al. 2003A (Roux, E.; Domloge, N.; Djabari, Z.; Dal Farra, C.; Bauza, E. 2003. Journal of Investigative Dermatology 121:1, 0500-0501) and Roux et al. 2003B (Roux, E.; Domloge, N.; Djabari, Z.; Dal Farra, C.; Bauza, E. 2003. Journal of Investigative Dermatology 121:1, 0505-0506). Their CAM-based system for investigating wound healing employed a method of interposition grafting where 3 mm punch biopsies of human skin were interposed gently in between the inner egg shell membrane and CAM of 7 day old chick embryo. Because human skin is interposed between the inner egg membrane and the CAM, there is no direct contact between the skin and the ambient atmosphere. Roux et al 2003A do not suggest the use of the CAM system to investigate wound healing in conjunction with topically applied factors, and indeed the lack of an epidermis-air interface could preclude such studies. There is no disclosure of any application other than wound healing studies in mechanically traumatized skin.
Transplantation of heterologous cells and tissues to the embryonic chick and, particularly, to the chorioallantoic membrane (CAM) is also being used to evaluate angiogenesis in human solid tumor growth and anti-neoplastic drug screening (see, for example, U.S. Pat. No. 6,228,345). The chick model system reproduces many of the characteristics of tumors in vivo, such as tumor mass formation, angiogenesis and metastasis. Only a few studies of human leukemia/lymphomas using this convenient system have been described, although none investigated whether engraftment to the hematopoetic system of the embryo took place. Longenecker, et al. (1977, J. Nat.l Cancer Inst. 58(4): 853-62), for example, examined whether human leukemic cells carry oncogenic viruses that attack the chicken, and not their engraftment and spread into the hematopoietic system.
Currently, the best in-vivo models for study of blood malignancy (and hematopoesis) are highly immune-deficient mice or fetal sheep and dogs (Cashman et al., 1997, Blood 89: 4307-4316; Zanjani, et al., 1992, Bone Marrow Transplant. 9 Suppl 1:86-9: 86-89; Lutzko et al., Exp Hematol. 2002(7):801-8). The fetal sheep or dog models are not practical for studying the biology of blood malignancies or for drug screening, because of the extremely high cost in money and space for the experiments. By contrast, NOD-SCID mice have been used extensively to study human blood malignancies. Large doses (5×107) of leukemia cells from patients are required for engraftment to irradiated NOD/SCID mice, and substantial engraftment takes six weeks to over two months (Dazzi et al., 2000, Hematol. J. 1: 307-315). When cells are grafted directly to the bone marrow (BM), a more technically demanding procedure with lower rates of survival, they engraft in 28 to 42 days, and human cells also home to in spleen in most mice (Yahata et al., 2003 Blood 101: 2905-2913). A more immune-deficient mouse model has been described in which homozygous disruption of the β2 microglobulin gene has been backcrossed onto the NOD/SCID background. These mice have higher levels of engraftment but are susceptible to spontaneous thymoma at an early age (6 months), reducing their value as a model for engraftment and drug testing for blood malignancies (Christianson et al., 1997, J. Immunol. 158: 3578-3586).
There exists a need for developing animal models for examining biological processes and screening drugs and therapeutic modalities on mammalian organs or tissues. Specifically, it would be highly advantageous to have a model system for large scale testing of biological processes related to mammalian cells and tissues affording physiological features such as immune cells and vasculature containing erythrocytes, while being readily accessible to manipulation and observation.